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Little of the life of Bp. Macarius is known. According to Eusebius of Caesarea, he became bishop of Jerusalem in 312. Bp. Macarius took part in the Council at Nicea in 325 during which he took a strong stand against the Arian formula, a position noted abusively by Arius in a letter to Eusebius of Nicomedia[1]. St. Athanasius, in one of his speeches against Arianism, refers to Macarius as an example of "the honest and simple style of apostolical men."

Bp. Macarius' name appears first among the bishops from Palestine who subscribed to the acts of the Council of Nicea; Eusebius of Caesarea's name appears fifth. At the council Macarius apparently contended with Eusebius of Caesarea concerning the rights of their respective sees as the seventh canon of the council noted, "As custom and ancient tradition show that the bishop of Aelia (Jerusalem) ought to be honored, he shall have precedence: without prejudice, however, to the dignity which belongs to the Metropolis," a vague statement that suggests a compromise.

St. Theophanes in his Chronography reported that, at the end of the Council at Nicea, the emperor Constantine I directed Bp. Macarius to search in Jerusalem for the sites of the Savior's Passion and Resurrection and for the True Cross. As confirmation of his direction, excavations in the city began shortly after the council and, contrary to expectations, the monument of our Savior's Resurrection was found under the remains of the temple of Venus. After hearing of the discovery, Constantine directed Macarius to erect a church (the Holy Sepulchre) on the site. Also about 325, St. Helen, the mother of Constantine, was accompanied by Bp. Macarius on her journey to Jerusalem, during which time she discovered the True Cross.

Sozomen [2] relates an attempt by Macarius to have a popular priest of Jerusalem, Maximus, installed as Bishop of Lydia (Diospolis), but Macarius' plan was forestalled by the people of Jerusalem, who did not want Maximus to leave their city. Maximus would eventual succeed Macarius to the see of Jerusalem.

Bp. Macarius most probably reposed in 334, as Maximus III, his successor, was present at the Council of Tyre in 335.